200
D. Cheng et al. / Journal of Fluorine Chemistry 128 (2007) 196–201
purification unless stated. Ethyl 4-(trimethylammonium)benzo-
ate trifluormethane-sulfonate 1 and N-succinimidyl-4-[(4-nitro-
reverse phase column and identified with reference compound
SFB, elution was performed at 1 mL/min with CH3CN/H2O
(1:1, v/v), tR = 4.25 min. Radio-TLC: CH2Cl2/EtOAc = 4:1,
Rf = 0.8.
benzensulfonyl)oxymethyl]benzoate
5
were synthesized
1
according to literatures [20,25], and confirmed by H NMR
spectral data (300 MHz, D2O) (compound 1): d 1.18–1.23 (3H,
t), 3.51 (9H, s), 4.20–4.27 (2H, q), 7.77–7.80 (2H, m), 8.04–
8.07 (2H, m); 1H NMR spectral data (300 MHz, CDCl3)
(compound 5): d = 2.92 (4H, s), 5.26 (2H, s), 7.45 (2H, d,
J = 1.33 Hz), 8.11 (4H, d, J = 2.59 Hz), 8.41 (2H, d,
J = 1.31 Hz). N-succinimidyl-4-fluorobenzoate (SFB) and N-
succinimidyl-4-(fluoromethyl) benzoate (SFMB) were synthe-
sized as the standard of [18F]SFB 4 and [18F]SFMB 6 referred to
literatures. 1H NMR (CDCl3) (SFB): d 2.89 (4H, s), 7.16–7.26
(2H, m), 8.13–8.18 (2H, m); 1H NMR (DMSO-d6) (SFMB): d
2.69 (4H, s), 5.17 (1H, s), 5.43 (1H, s), 7.52–7.61 (2H, m),
8.08–8.13 (2H, m) [26,27].
4.4. Synthesis of [18F]SFMB 6
N-succinimidyl 4-[(nitrobenzenslfonyl)oxymethyl]benzo-
ate 5 (2 mg in 200 mL anhydrous CH3CN) was added to the
vial containing the dried Kryptofix12.2.2/K+ complex of
[18F]Fꢁ and reacted at 80 8C for 10 min. After cooling for
2 min, the solution was diluted with 2 mL CH2Cl2/Hexane
(1:1, v/v) and loaded onto an activated Sep-Pak silica cartridge
(Waters, Sep-Pak Plus). Then N-succinimidyl-4-[18F] (fluor-
omethyl)-benzoate 6 ([18F]SFMB) was eluted with 2 mL
CH2Cl2. The compound 6 was analyzed by radio-HPLC using
reverse phase column and identified with reference compound
SFMB, elution was performed at 1 mL/min with CH3CN/H2O
(1:1, v/v), tR = 4.28 min. Radio-TLC: CH2Cl2/EtOAc = 4:1,
Rf = 0.9.
High-performance liquid chromatography (HPLC) was
carried out on a system consisting of a P680pump, a PDA-
100 photodiode Array Detector and a NaI (Tl) scintillation
detector with the column being LiChrosorb C18 (10 mm,
300 mm ꢂ 3.9 mm). Radio-TLC analyses were performed
using silica gel 60 GF-254 plates (Merck), a Bioscan system
AR-2000 and Winscan software, version 3.09. 1H spectra were
obtained using a Bruker AM-300 spectrometer (300 MHz).
4.5. Conjugation of [R8,15,21, L17]-VIP with [18F]SFB 4
and [18F]SFMB 6
In a v-vial containing the 100 mg [R8,15,21, L17]-VIP peptide
in borate buffer (200 mL, 0.1 mol/L, pH 8.0), [18F]SFB 4 in
50 mL CH3CN was added. The reaction mixture was kept at
room temperature for 20 min. The labeling efficiency was
determined by radio-TLC (eluent: CH3OH/H2O = 8:3, v/v,
Rf = 0.1). The product was purified by radio-HPLC, which was
performed at 1 mL/min with a gradient of 0.1% TFA in CH3CN
(solvent A) and 0.1% TFA in water (solvent B) as the following:
0–12 min, 75% B; 12–32 min, 75–30% B. Fraction 1
(tR = 11.43 min) was collected as [18F]FB-[R8,15,21, L17]-VIP
7. Synthesis of [18F]FMB-[R8,15,21, L17]-VIP 8 was operated in
a similar procedure as above, only pH was changed to 8.7, and
fraction 2 of the product (tR = 12.23 min) was collected.
[18F]FB-[R8,15,21, L17]-VIP 7 and [18F]FMB-[R8,15,21, L17]-VIP
8 were analyzed by radio-HPLC with over 99% of the
radiochemical purity.
4.2. Production of [18F]fluoride
The anhydrous [18F]fluoride was obtained from the mixture
of Kryptofix12.2.2 (1–2 mg) and K2CO3 (10–12 mg) solution
azeotropically removing water with 0.5 mL acetonitrile for 3
times in a stream of N2.
4.3. Synthesis of [18F]SFB 4
Ethyl 4-(trimethylammonium)benzoate trifluormethane-sul-
fonate 1 (10 mg in 200 mL anhydrous CH3CN) was added to the
vial containing the dried Kryptofix12.2.2/K+ complex of
[18F]Fꢁ and reacted at 90 8C for 5 min.
Then the ethyl ester 2 was hydrolyzed using 0.5 mL 1 mol/
L NaOH at 90 8C for 5 min, and acidified with 0.8 mL 1 mol/L
HCl. The solution was diluted with 2 mL H2O and loaded onto
an activated C18 Sep-Pak1 cartridge (Waters, Sep-Pak1
Plus). The Sep-Pak1 cartridge was washed with 2 mL
0.1 mol/L HCl, then a MgSO4 column was connected at the
end of the cartridge and the 4-[18F]fluorobenzoic acid 3
([18F]FBA) was eluted with 2 mL CH3CN. Radio-TLC
revealed radiochemical purity of over 98% (eluent: CH2Cl2/
EtOAc = 4:1, v/v, Rf = 0.2). Afterwards, tetrapropylammo-
nium hydroxide (15 mL, 1 M in H2O), O-(N-succinimidyl)
N,N,N0,N0-tetramethyluronium tetrafluoroborate (10 mg) were
added to the vial containing [18F]FBA 3 (CH3CN solution) and
reacted at 90 8C for 5 min. Instantaneous acidification was
performed with 3 mL of 5% HOAc and diluted with 6 mL
H2O. The mixture was loaded onto an activated C18 Sep-Pak1
cartridge. The cartridge was washed with 10 mL CH3CN/H2O
(1:7, v/v) and purified [18F]SFB 4 was eluted with 2 mL
CH2Cl2. The product was analyzed by Radio-HPLC using
4.6. Stability test
To measure the in vitro stability, [18F]FB-[R8,15,21, L17]-VIP
7 and [18F]FMB-[R8,15,21, L17]-VIP 8 were incubated at 37 8C
in HSA, respectively. At the end of incubation, samples were
analyzed by radio-HPLC and the same operation was carried
out at different time points till 4 h.
In order to evaluate defluorination stability of the products
in vivo, 20 mCi of [18F]FB-[R8,15,21, L17]-VIP 7 in 200 mL
phosphate-buffered saline was injected to BALB/c mice
intravenously. One hour post injection mice (n = 5) were
sacrificed and bones were isolated for weighing and
counting. The bones radioactivity were determined as
percent injected dose per gram tissue (% ID/g). The same
operations were carried out with [18F]FMB-[R8,15,21, L17]-
VIP 8.
Cheng, Dengfeng
